Optimization and validation of PIKFYVE antagonism as a therapy for C9ORF72-ALS/FTD Project Summary / Abstract The C9ORF72 repeat expansion mutation is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), accounting for over 50% of ALS cases in northern Europe and 10% of cases worldwide, making it a critical target for therapeutic intervention. Using patient-specific stem cell-based disease models, animal models, and postmortem tissue analysis, we have identified a new therapeutic target for C9ORF72 ALS/FTD, the lipid kinase PIKFYVE. We find that inhibition of PIKFYVE rescues the endosomal trafficking defects in C9ORF72 motor neurons and restores normal motor neuron survival. PIKFYVE functions in a manner that opposes FIG4, a phosphatase for which a loss-of-function mutation causes ALS. Antisense oligonucleotide-mediated knockdown of PIKFYVE rescues C9-ALS motor neuron survival, without exhibiting any toxicity toward control motor neurons. This combined functional and genetic evidence strongly indicate that small molecule inhibition of PIKFYVE kinase is a viable therapeutic target for C9-ALS/FTD. We have found that Apilimod reverses survival and other functional defects and is an effective PIKFYVE small molecule inhibitor. Apilimod has been tested in the clinic where target engagement without patient toxicity was observed, and we?ve determined it to be well tolerated in mice. We have begun execution of a two-pronged strategy to generate novel inhibitors of PIKFYVE. The first approach focuses on rescaffolding Apilimod to create a small molecule that can cross the BBB and can be patented. As a backup, our second approach employs virtual screening to identify new, patentable chemotypes which inhibit PIKFYVE. To this end, we have constructed 3D homology protein models for human PIKFYVE which we?ve used productively in rescaffolding to generate several Apilimod analogs. We employed the PIKFYVE homology models and the Small Molecule Drug Discovery Suite from Schrodinger and screened over 8 million compounds available virtually from the Icagen and E-molecule electronic compound collections. To develop structure activity relationships (SAR) for this program, we have established a biochemical PIKFYVE kinase assay as the primary assay for all compound evaluations. The goal of this Fast Track project is to identify a potent PIKFYVE inhibitor that is blood-brain-barrier penetrating. In phase I, we will use Apilimod, the 6 analogs from Table 1 and the 3 most promising leads from the in silico screen to test our entire funnel, including in vivo assays. In Phase II we will use these assays to optimize and validate a development candidate. Our specific aims are (Phase I) 1) Validation of primary and secondary in vitro assays; 2) Validation of tertiary assays; 3) Validation of proof of concept assays; (Phase II) 1) Compound optimization in primary and secondary assays; 2) Optimize of compound safety and administration through tertiary assays; 3) Establish in vivo proof of concept in C9ORF72 ALS/FTD.